Stanislav Chladek

Stimulation of Escherichia Coli elongation factor Tu-dependent GTP hydrolysis by aminoacyl oligonucleotides in the presence of aurodox

During protein synthesis in a ~rocaryotjc e&l, elongation factor Tu plays an important role by promoting the binding of AA-tRNA to ribosomes. This binding is mediated through the formation of a ternary complex of AA-tRNA . EF-Tu . GTP, followed by the hydrolysis of CTP during the binding of AA-tRNA to the ribosomal A site. The binary complex EF-Tu GDP is then released from the ribosome and is recycled through the formation of transient EF-Tu Ts complex. After the displacement of EF-Ts by GTP, the EF-Tu . GTP can interact with another molecule af AAtRNA [3], The antibiotic ~rromy~in (or its derivatives, e.g., Aurodox) inhibits protein biosynthesis by virtue of its interaction with EF-Tu and effects practically all the EF-Tu-dependent reactions, presumably through a conformati~nal alteration of EF-Tu [4,5]. This antibiotic enables EF-Tu alone to catalyze the hydrolysis of GTP in the absence of other com~nents such as AA-tRNA and ribosomes which are otherwise required.

Inhibition of the peptidyl transferase A-site function by 2'-O-aminoacyloligonucleotides.

Abstract New “non-isomerizable” analogs of the 3′-terminus of AA-tRNA, C-A(2′Phe)H, C-A(2′Phe)Me, C-A(2′H)Phe and C-A(2′Me)Phe, were tested as acceptor substrates of ribosomal peptidyl transferase and inhibitors of the peptidyl transferase A-site function. The 3′-O-AA-derivatives were active acceptors of Ac-Phe in the peptidyl transferase reaction, while the 2′-O-AA-derivatives were completely inactive. Both 2′- and 3′-O-AA-derivatives were potent inhibitors of peptidyl transferase catalyzed Ac-Phe transfer to puromycin. The results indicate that although peptidyl transferase exclusively utilizes 3′-O-esters of tRNA as acceptor substrates, its A-site can also recognize the 3′-terminus of 2′-O-AA-tRNA.

The role of the cytidine residues of the tRNA 3′-terminus at the peptidyltransferase A- and P-sites

The 3’-terminal nucleotides of tRNA play a major role in the interaction of both aminoacyl and peptidyl-tRNAs with the acceptor and donor sites of peptidyltransferase [ 11. Although the minimal requirement for donor activity is the presence of 2’(3’)-O-(N-acyl)aminoacyl)adenosine 5’-phosphate [2] at the donor site, and 2’(3’)-O-aminoacyladenosine is a minimal acceptor substrate [3], the activity of these simple models in the peptidyltransferase reaction is considerably increased by adding either a cytidine or a cytidine 3’-phosphate residue, respectively. Thus, C-A(fMet) is -30-times more active as a donor substrate than pA(fMet) [4], and C-C-A(fMet) is still more active [5]. At the acceptor site, C-A-Gly is a good acceptor substrate, whereas A-Gly has no activity [6], and C-A-Phe is -5OO-times more active than A-Phe [7]. Cytidine 5’-phosphate strongly stimulates the donor activity of, e.g., pA(fMet), most probably by occupying that part of the donor site of peptidyltransferase which would otherwise bind the penultimate cytidine residue of the 3’-terminus of peptidyltRNA, thus simulating the presence of, e.g., pC-A(fMet) [8]. In contrast, the transfer of AcPhe residue from AcPhe-tRNA to puromycin is not significantly influenced by Cp, pC and C-C [9]. Despite this evidence several reports have claimed that the cytidylic acid residues of the acceptor sub-

Possible relationship of peptidyl transferase binding sites, 5S RNA and peptidyl-tRNA

Abstract A possible role of the 5S RNA in the peptide chain elongation process is suggested. This role consists of an interaction of the ψ loop of peptidyl tRNA at the P-site of peptidyl transferase with the single stranded region of the 5S RNA. The interacting region may also be one of the sites for peptidyl transferase binding to the 5S RNA, moreover, the resulting “release” of peptidyl transferase from its binding site on the 5S RNA (due to binding of the ψ loop of peptidyl tRNA to the 5S RNA) could cause conformational changes in the enzyme leading to an exposure of its “CCA binding site” for peptidyl tRNA.

The synthesis of 5′ thioanalogs of polydeoxyribonucleotides

Abstract Oligo 5′-thiodeoxythymidylates and oligo 5′-thiodeoxyadenylates were prepared via displacement of tosylates by thiophosphate mono- and diesters. The 5′ ends contain O-tosyl or S-phosphoryl groups, while the 3′ ends terminate in deoxythymidine. The types of starting materials used for such polymerization were 5′-O-tosyl nucleoside 3′ cyanoethyl phosphorothionates and the corresponding monoesters. Both oligonucleotide analogs possess secondary structure according to spectral evidence.

New Approach to the Synthesis of 2′(3′)-O-Aminoacyloligoribonucleotides Related to the 3′-Terminus of Aminoacyl Transfer Ribonucleic Acid

Abstract A new methodology for the synthesis of 2′(3′)-0-aminoacyl oligonucleotides based on an unique combination of protecting groups is described. The blocking scheme allows a simple two step deblocking procedure, which provides easy access to the target compounds.

General Synthesis of 2′(3′)-O-Aminoacyl Oligoribonucleotides Related to the 3′-Terminus of aa-tRNA

Abstract A general method is presented for the synthesis of the title compounds using phosphotriester methodology and employing a unique combination of protecting groups, including the double protection of the guanosine aglycon.